Mishin 2010 Free Radical Biol Med: Difference between revisions
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{{Publication | {{Publication | ||
|title=Mishin V, Gray JP, Heck DE, Laskin DL, Laskin JD (2010) Application of Amplex red/horseradish peroxidase assay to measure hydrogen peroxide production by recombinant microsomal enzymes. Free | |title=Mishin V, Gray JP, Heck DE, Laskin DL, Laskin JD (2010) Application of Amplex red/horseradish peroxidase assay to measure hydrogen peroxide production by recombinant microsomal enzymes. Free Radic Biol Med 48:1485-91. | ||
|info=[http://www.ncbi.nlm.nih.gov/pubmed/20188819 PMID: 20188819 Open access] | |||
|authors=Mishin V, Gray JP, Heck DE, Laskin DL, Laskin JD | |authors=Mishin V, Gray JP, Heck DE, Laskin DL, Laskin JD | ||
|year=2010 | |year=2010 | ||
|journal=Free | |journal=Free Radic Biol Med | ||
|abstract=The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species. | |||
|keywords=[[Amplex red]], [[Horseradish peroxidase]], [[Hydrogen peroxide]] | |keywords=[[Amplex red]], [[Horseradish peroxidase]], [[Hydrogen peroxide]] | ||
}} | }} | ||
{{Labeling | {{Labeling | ||
| | |area=Instruments;methods | ||
|preparations=Enzyme | |preparations=Enzyme | ||
|injuries=Oxidative stress;RONS | |||
}} | }} | ||
Latest revision as of 16:40, 23 February 2015
| Mishin V, Gray JP, Heck DE, Laskin DL, Laskin JD (2010) Application of Amplex red/horseradish peroxidase assay to measure hydrogen peroxide production by recombinant microsomal enzymes. Free Radic Biol Med 48:1485-91. |
Mishin V, Gray JP, Heck DE, Laskin DL, Laskin JD (2010) Free Radic Biol Med
Abstract: The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species. • Keywords: Amplex red, Horseradish peroxidase, Hydrogen peroxide
Labels: MiParea: Instruments;methods
Stress:Oxidative stress;RONS
Preparation: Enzyme
